UNIPROT:P17174 - FACTA Search

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Query: UNIPROT:P17174 (aspartate aminotransferase)
14,872 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tyrosine-225 is hydrogen-bonded to the 3'-hydroxyl group of pyridoxal 5'-phosphate in the active site of aspartate aminotransferase. Replacement of this residue with phenylalanine (Y225F) results in a shift in the acidic limb of the pKa of the kcat/KAsp vs pH profile from 7.1 (wild-type) to 8.4 (mutant). The change in the kinetic pKa is mirrored by a similar shift in the spectrophotometrically determined pKa of the protonated internal aldimine. Thus, a major role of tyrosine-225 is to provide a hydrogen bond that stabilizes the reactive unprotonated form of the internal aldimine in the neutral pH range. The Km value for L-aspartate and the dissociation constant for alpha-methyl-DL-aspartate are respectively 20- and 37-fold lower in the mutant than in the wild-type enzyme, while the dissociation constant for maleate is much less perturbed. These results are interpreted in terms of competition between the Tyr225 hydroxyl group and the substrate or quasi-substrate amino group for the coenzyme. The value of kcat in Y225F is 450-fold less than the corresponding rate constant in wild type. The increased affinity of the mutant enzyme for substrates, combined with the lack of discrimination against deuterium in the C alpha position of L-aspartate in Y225F-catalyzed transamination [Kirsch, J. F., Toney, M. D., & Goldberg, J. M. (1990) in Protein and Pharmaceutical Engineering (Craik, C. S., Fletterick, R., Matthews, C. R., & Wells, J., Eds.) pp 105-118, Wiley-Liss, New York], suggests that the rate-determining step in the mutant is hydrolysis of the ketimine intermediate rather than C alpha-H abstraction which is partially rate-determining in wild type.
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PMID:The tyrosine-225 to phenylalanine mutation of Escherichia coli aspartate aminotransferase results in an alkaline transition in the spectrophotometric and kinetic pKa values and reduced values of both kcat and Km. 198 27

D- and L-aminooxysuccinate were synthesized and evaluated as inhibitors of cytoplasmic aspartate aminotransferase (EC 2.6.1.1) from porcine heart. L-Aminooxysuccinate was shown to be a slow binding inhibitor of the pyridoxal phosphate form of the enzyme with a Ki of 160 nM and a half-life of the inhibited complex of 8 min. Kinetic analysis revealed that inhibition followed a two-step mechanism in which the last step was rate-limiting. D-Aminooxysuccinate was not inhibitory up to a concentration of 0.1 mM. These compounds were compared to D- and L-hydrazinosuccinate, which are potent slow binding inhibitors of aspartate aminotransferase with Ki values of 1.5 and 0.5 nM, respectively. Models of all four analogs were built into the active site of the closed form of the enzyme. The energy-minimized conformations of both L-isomers bound to aspartate aminotransferase show better geometry for hydrogen bond and ion pair formation than do the corresponding D-isomers. The aldimine double bond formed by the L-isomers is not coplanar with the pyridoxal phosphate ring in accordance with the spectral properties of the inhibitor complexes that are characterized by broad absorbance bands. This lack of planarity was not evident for the models of D-hydrazinosuccinate and D-aminooxysuccinate.
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PMID:Inhibition of cytoplasmic aspartate aminotransferase from porcine heart by R and S isomers of aminooxysuccinate and hydrazinosuccinate. 200 95

In aspartate aminotransferase (AspAT), His143 is located within a hydrogen-bonding distance to Asp222 that forms a strong ion pair with the ring nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP). His143 of Escherichia coli AspAT was replaced by Ala or Asn. The mutant enzyme H143A showed a slight increase in the maximum velocity of the overall transamination reaction between aspartate and 2-oxoglutarate, while H143N AspAT showed a decrease to 60% in the maximum rate of the overall reactions in both directions. In all of the half-transamination reactions with four substrates, aspartate, glutamate, oxalacetate, and 2-oxoglutarate, the catalytic competence as defined by kmax/Kd decreased by 3-18-fold upon replacing His143 by either Ala or Asn. The extent of the decrease varied from one substrate to another; it was largely contributed to by the decrease in affinities for all substrates. The equilibrium constants, [PMP-form] [keto acid]/[( PLP-form] [amino acid]), decreased by over 10-fold upon the mutations at position 143. Both H143A and H143N AspATs exhibited a considerably decreased affinity for 2-methylaspartate, an external-aldimine-forming substrate analogue, yet without appreciable alteration in the affinity for succinate and glutarate, which are non-aldimine-forming analogues. All these findings suggest that, although His143 is not essential for catalysis, it might assist the formation of enzyme-substrate complex.
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PMID:The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase. 200 66

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.
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PMID:Complete amino acid sequence of rat liver cytosolic alanine aminotransferase. 204 42

X-Ray structural data concerning the substrate binding site of cytosolic chicken aspartate aminotransferase (AspAT) are reported. The structure of the complex of AspAT with the substrate-like inhibitor maleate has been refined at 2.2 A resolution. The lengths of hydrogen bonds between a bound molecule of maleate and side chains of amino acid residues in the active site are presented as well as other interatomic distances in the substrate binding site. The data obtained for the cytosolic AspAT have been compared with those for the mitochondrial chicken AspAT. It has been inferred that differences in substrate specificity of the AspAT isoenzymes are determined by interactions involving amino acid residues which are situated in the immediate vicinity of the active site and influence ionization or orientation of functional groups interacting with substrate. An explanation is suggested for different rates of transamination of aromatic amino acids in the active sites of the cytosolic and mitochondrial isoenzymes.
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PMID:[Structural basis for differences in substrate specificity of aspartate aminotransferase isoenzymes]. 234 26

The C alpha primary hydrogen kinetic isotope effects (C alpha-KIEs) for the reaction of the cytoplasmic isozyme of aspartate aminotransferase (cAATase) with [alpha-2H]-L-aspartate are small and only slightly affected by deuterium oxide solvent (DV = 1.43 +/- 0.03 and DV/KAsp = 1.36 +/- 0.04 in H2O; DV = 1.44 +/- 0.01 and DV/KAsp = 1.61 +/- 0.06 in D2O). The D2O solvent KIEs (SKIEs) are somewhat larger and are essentially independent of deuterium at C alpha (D2OV = 2.21 +/- 0.07 and D2OV/KAsp = 1.70 +/- 0.03 with [alpha-1H]-L-aspartate; D2OV = 2.34 +/- 0.12 and D2OV/KAsp = 1.82 +/- 0.06 with [alpha-2H]-L- aspartate). The C alpha-KIEs on V and on V/KAsp are independent of pH from pH 5.0 to pH 10.0. These results support a rate-determining concerted 1,3 prototropic shift mechanism by the multiple KIE criteria [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. The large C alpha-KIEs for the reaction of mitochondrial AATase (mAATase) with L-glutamate (DV = 1.88 +/- 0.13 and DV/KGlu = 3.80 +/- 0.43 in H2O; DV = 1.57 +/- 0.05 and DV/KGlu = 4.21 +/- 0.19 in D2O) coupled with the relatively small SKIEs (D2OV = 1.58 +/- 0.04 and D2OV/KGlu = 1.25 +/- 0.05 with [alpha-1H]-L-glutamate; D2OV = 1.46 +/- 0.06 and D2OV/KGlu = 1.16 +/- 0.05 with [alpha-2H]-L-glutamate) are most consistent with a two-step mechanism for the 1,3 prototropic shift for this isozyme-substrate pair.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic isotope effect studies on aspartate aminotransferase: evidence for a concerted 1,3 prototropic shift mechanism for the cytoplasmic isozyme and L-aspartate and dichotomy in mechanism. 254 82

Resonance Raman (RR) spectra are reported for aspartate aminotransferase from pig heart cytosol, and for inhibitor complexes. They are interpreted with reference to the previously analyzed spectra of pyridoxal phosphate (PLP) Schiff base adducts. This comparison shows that, as expected, the pyridine N atom is protonated in the native enzyme at pH 5, and in the glutarate complexes at pH 8.5, and that it is also protonated in the alpha-methylaspartate complex; the stabilization of the pyridine proton at high pH must be due to the interaction with aspartate 222 seen in the x-ray crystal structure. RR spectra of the erythro-beta-hydroxy-DL-aspartate complex, representing the p-quinoid enzyme intermediate, as well as of AlIII complexes of PLP Schiff bases with phenylalanine and tyrosine ethyl ester have been obtained via the coherent anti-Stokes Raman scattering technique, and partially assigned. A novel H/D exchange at the coenzyme C4' atom has been observed for the native enzyme in D2O, and has been determined, by a combination of NMR and RR measurements, to be due to the Raman laser irradiation. This photoprocess, which is not observed for PLP Schiff bases in aqueous solution, is attributed to a photoexcited p-quinoid intermediate, similar to that implicated in the enzyme mechanism. It is suggested that this intermediate is stabilized by protein interactions which localize charge on the phenolate O atom, plausibly a hydrogen bond from the nearby tyrosine 225. H/D exchange would then follow via the aldimine-ketimine interconversion known to take place in the enzyme reaction.
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PMID:Resonance Raman spectra of the pyridoxal coenzyme in aspartate aminotransferase. Evidence for pyridine protonation and a novel photochemical H/D exchange at the imine carbon atom. 299 44

The pharmacology, pharmacokinetics, clinical efficacy, adverse effects, and dosage and administration of omeprazole are reviewed. Omeprazole, a substituted benzimidazole, has a unique site and mechanism of action because it inhibits the proton pump--i.e., hydrogen, potassium adenosine triphosphatase (H+,K+-ATPase)--and consequently blocks the final common step in the gastric acid secretory pathway. Omeprazole inhibits basal and histamine-, gastrin- and pentagastrin-stimulated gastric hydrochloric acid secretion. It produces a dose-dependent reduction in gastric acidity, gastric acid output, and gastric juice volume and has variable effects on pepsin secretion. Omeprazole has no documented effect on esophageal motility or lower esophageal sphincter pressure. Omeprazole is variably absorbed from the gastrointestinal tract, and food appears to decrease the rate, but not the extent, of drug absorption. The drug is approximately 95% bound to plasma proteins and is metabolized to inactive components that are enterohepatically or renally eliminated. Omeprazole is more effective (in most studies) than H2-receptor antagonists in treating duodenal ulcer, at least as effective in treating benign gastric ulcer, and more effective in treating reflux esophagitis. Omeprazole has been used successfully in patients with Zollinger-Ellison syndrome refractory to treatment with H2-receptor antagonists. Gastrointestinal complaints (nausea and diarrhea) are the most commonly reported adverse effects associated with omeprazole therapy. The most frequently reported laboratory abnormality occurring with omeprazole use is elevation of serum aspartate aminotransferase and alanine aminotransferase concentrations. Omeprazole will serve a valuable role in the management of gastrointestinal tract ulcers and hypersecretory conditions.
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PMID:Therapeutic evaluation of omeprazole. 306 85

The crucial step in enzymatic transamination is the tautomerization of aldimine/ketimine intermediates, formed between the pyridoxyl coenzyme and the amino/keto acid substrate, which is catalyzed primarily by the active site residue Lys-258 (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921; W. L. Finlayson and J. F. Kirsch, in preparation). Tyr-70 is localized in close proximity to Lys-258 and, in addition, forms a hydrogen bond with the coenzyme phosphate. Tyr-70 has been postulated to have an important role in the tautomerization (Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525). This hypothesis has now been tested by the construction and analysis of a mutant Escherichia coli aspartate aminotransferase in which Tyr-70 has been changed to Phe (Y70F). Y70F retains at least 15% of the maximal activity of the wild type enzyme (WT) (kcat = 170 +/- 15 s-1 for WT versus greater than or equal to 26 +/- 3 s-1 for Y70F and shows increased Michaelis constants for both substrates (KmAsp = 2.5 +/- 0.4 mM; Km alpha Kg = 0.59 +/- 0.08 mM for WT versus KmAsp = 3.9 +/- 0.3 mM; Km alpha Kg = 2.70 +/- 0.02 mM for Y70F (where alpha Kg is alpha-ketoglutarate) ). The spectrophotometrically determined pK a values of the internal aldimines formed between pyridoxal 5'-phosphate (PLP) and Lys-258 are identical for WT and Y70F. In assays where excess L-aspartate and excess PLP are incubated with either WT or Y70F, the mutant enzyme converts the free PLP to free pyridoxamine 5'-phosphate 80-fold faster than WT (k = (3.75 +/- 0.23) X 10(-2)s-1 for Y70F versus (4.90 +/- 0.02) X 10(-4)s-1 for WT). Y70F also converts free pyridoxamine 5'-phosphate to free PLP faster than WT. Thus, Y70F dissociates coenzyme more readily than does WT. It therefore appears that the role of Tyr-70 is mainly in preventing the dissociation of the coenzyme from the enzyme. Tyr-70 does not function in an essential chemical step.
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PMID:Tyrosine 70 increases the coenzyme affinity of aspartate aminotransferase. A site-directed mutagenesis study. 330 7

The aim of this study was tracing of changes in the activity of glutathione peroxidase (GSHPx), glutathione transferase (GSH S-Tr), aspartate aminotransferase (AspAT) and alanine aminotransferase (A1AT) in the brain as a result of diet enrichment with antioxidants: selenium (Se), vitamin E and vitamin B15 (pangamic acid). The experiment was carried out on Wistar rats with initial body weight 150 g. Following prolonged enrichment of diet with Se (0.1 ppm of sodium selenite), vitamin E (6 mg/100 g of diet) and vitamin B15 (2.5 mg/100 g of diet) the following results were obtained. The activity of GSHPx in brain microsomes was not changed after one year of vitamin E administration when it was measured against hydrogen hydroxide and against cumene hydrochloride; vitamin E administration increased the activity of GSH S-Tr in the cytoplasmic fraction of brain cells. Diet enrichment with selenium increased after 12 and 18 months the activity of GSHPx measured against both substrates, and GSH S-Tr activity increased considerably. Presence of vitamin B15 in diet reduced GSHPx activity after one-year or longer administration, after 18 months the activity of GSH S-Tr was reduced also. No changes were noted in the activity of AspAT and A1AT.
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PMID:The effect of long-term enrichment of diet with selenium, vitamin E and B15 on the activity of certain enzymes in rat brain. 345 69

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